Isolation and characterization of plant growth-promoting rhizobacteria and their effects on the growth of Medicago sativa L. under salinity conditions

Abstract

Plant growth-promoting rhizobacteria are a group of free-living bacteria that colonize plant rhizosphere and benefit plant root growth, thereby increasing host plant to cope with salinity induced stress. The aim of this study was to (1) isolate and characterize auxin-producing bacteria showing a high plant growth-promoting (PGP) potential, and (2) evaluate the PGP effects on the growth of Medicago sativa L under salinity stress (130 mM NaCl). Of thirteen isolates, Bacillus megaterium NRCB001 (NRCB001), B. subtilis subsp. subtilis NRCB002 (NRCB002) and B. subtilis NRCB003 (NRCB003) had the ability to produce auxin, which ranged from 47.53 to 154.38 μg ml−1. The three auxin-producing bacterial strains were shown multiple PGP traits, such as producing siderophore and NH3, showing ACC deaminase activity, solubilize phosphate and potassium. Furthermore, NRCB001, NRCB002, and NRCB003 could survive in LB medium containing 1750 mM NaCl. The three auxin-producing with salinity tolerance strains were selected for further analyses. In greenhouse experiments, when inoculated with NRCB001, NRCB002 and NRCB003, dry weight of alfalfa significantly (P < 0.05) increased by 24.1%, 23.1% and 38.5% respectively, compared with those of non-inoculated control seedlings under normal growth condition. When inoculated with NRCB002 and NRCB003, dry weight of alfalfa significantly (P < 0.05) increased by 96.9 and 71.6% respectively, compared with those of non-inoculated control seedlings under 130 mM NaCl condition. Our results indicated that NRCB002 and NRCB003 having PGP traits are promising candidate strains to develop biofertilizers, especially used under salinity stress conditions.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Aslam F, Ali B (2018) Halotolerant bacterial diversity associated with suaeda fruticose (L.) forssk. improved growth of maize under salinity stress. Agronomy 8(8):131

    CAS  Google Scholar 

  2. Baldani JI, Reis VM, Videira SS, Boddey LH, Baldani VLD (2014) The art of isolating nitrogen-fixing bacteria from non-leguminous plants using N-free semi-solid media: a practical guide for microbiologists. Plant Soil 384:413–431

    CAS  Google Scholar 

  3. Bric JM, Bostock RM, Silverstone SE (1991) Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl Environ Microbiol 57:535–538

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Chun J, Bae KS (2000) Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences. Antonie Van Leeuwenhoek 78:123–127

    CAS  PubMed  Google Scholar 

  5. Daur I, Saad MM, Eida AA, Ahmad S, Shah ZH, Ihsan MZ, Muhammad Y, Sohrab SS, Hirt H (2018) Boosting alfalfa (Medicago sativa L.) production with rhizobacteria from various plants in saudi arabia. Front Microbiol 9:447

    Google Scholar 

  6. Dimkpa C, Weinand T, Asch F (2009) Plant–rhizobacteria interactions alleviate abiotic stress conditions. Plant Cell Environ 32:1682–1694

    CAS  PubMed  Google Scholar 

  7. Egamberdieva D, Davranov K, Wirth S, Hashem A, Abd Allah EF (2017) Impact of soil salinity on the plant-growth-promoting and biological control abilities of root associated bacteria. Saudi J Biol Sci 24(7):1601–1608

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Etesami H, Beattie GA (2018) Mining halophytes for plant growth-promoting halotolerant bacteria to enhance the salinity tolerance of non-halophytic crops. Front Microbiol 9:148

    PubMed  PubMed Central  Google Scholar 

  9. Etesami H, Maheshwari DK (2018) Use of plant growth promoting rhizobacteria (PGPRs) with multiple plant growth promoting traits in stress agriculture: action mechanisms and future prospects. Ecotox Environ Safe 156:225–246

    CAS  Google Scholar 

  10. Etesami H, Alikhani HA, Hosseini HM (2015) Indole-3-acetic acid (IAA) production trait, a useful screening to select endophytic and rhizosphere competent bacteria for rice growth promoting agents. MethodsX 2:72–78

    PubMed  PubMed Central  Google Scholar 

  11. Ferreira NC, Mazzuchelli RDCL, Pacheco AC, Araujo FFD, Antunes JEL, Araujo ASFD (2018) Bacillus subtilis improves maize tolerance to salinity. Cienc Rural 48:8

    Google Scholar 

  12. Gao N, Shen WS, Camargo E, Shiratori Y, Nishizawa T, Isobe K, He XH, Senoo K (2017) Nitrous oxide (N2O)-reducing denitrifier-inoculated organic fertilizer mitigates N2O emissions from agricultural soils. Biol Fertil Soils 53:885–898

    CAS  Google Scholar 

  13. Glick BR (2014) Bacteria with ACC deaminase can promote plant growth and help to feed the world. Microbiol Res 169:30–39

    CAS  PubMed  Google Scholar 

  14. Gupta M, Kiran S, Gulati A, Singh B, Tewari R (2012) Isolation and identification of phosphate solubilizing bacteria able to enhance the growth and aloin-A biosynthesis of Aloe barbadensis Miller. Microbiol Res 167:358–363

    CAS  Google Scholar 

  15. Hashem A, Tabassum B, Abd Allah EF (2019) Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress. Saudi J Biol Sci 26:1291–1297

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Hmaeid N, Wali M, Metoui-Ben Mahmoud O, Pueyo JJ, Ghnaya T, Abdelly C (2019) Efficient rhizobacteria promote growth and alleviate NaCl-induced stress in the plant species Sulla carnosa. Appl Soil Ecol 133:104–113

    Google Scholar 

  17. Hongoh Y, Ohkuma M, Kudo T (2003) Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera; Rhinotermitidae). FEMS Microbiol Ecol 44:231–242

    CAS  PubMed  Google Scholar 

  18. Hu XF, Chen JS, Guo JF (2006) Two phosphate- and potassium-solubilizing bacteria isolated from Tianmu Mountain, Zhejiang, China. World J Microb Biot 22:983–990

    CAS  Google Scholar 

  19. Hussein KA, Joo JH (2015) Isolation and characterization of rhizomicrobial isolates for phosphate solubilization and indole acetic acid production. J Korean Soc Appl Biol Chem 58:847–855

    CAS  Google Scholar 

  20. Jin YQ, Zhu HF, Luo S, Yang WW, Zhang L, Li SS, Jin Q, Cao Q, Sun S, Xiao M (2019) Role of maize root exudates in promotion of colonization of bacillus velezensis strain S3-1 in rhizosphere soil and root tissue. Curr Microbiol 76:855–862

    CAS  PubMed  Google Scholar 

  21. Kadmirl IM, Chaouqul L, Azaroual SE, Sijilmassl B, Yaakoubl K, Wahby I (2018) Phosphate-solubilizing and IAA-producing rhizobacteria promote plant growth under saline conditions. Arab J Sci Eng 43:3403–3415

    Google Scholar 

  22. Karthik C, Elangovan N, Kumar TS, Govindharaju S, Barathi S, Oves M, Arulselvi PI (2017) Characterization of multifarious plant growth promoting traits of rhizobacterial strain AR6 under Chromium (VI) stress. Microbiol Res 204:65–71

    CAS  PubMed  Google Scholar 

  23. Khan WU, Ahmad SR, Yasin NA, Ali A, Ahmad A, Akram W (2017) Application of Bacillus megaterium MCR-8 improved phytoextraction and stress alleviation of nickel in Vinca rosea. Int J Phytorem 19(9):813–824

    CAS  Google Scholar 

  24. Li HQ, Jiang XW (2017) Inoculation with plant growth-promoting bacteria (PGPB) improves salt tolerance of maize seedling. Russ J Plant Physiol 64(2):235–241

    CAS  Google Scholar 

  25. Liu ZH, Zhang HM, Li GL, Guo XL, Chen SY, Liu GB, Zhang YM (2011) Enhancement of salt tolerance in alfalfa transformed with the gene encoding for betaine aldehyde dehydrogenase. Euphytica 178:363–372

    CAS  Google Scholar 

  26. Liu JL, Tang L, Gao H, Zhang M, Guo C (2019) Enhancement of alfalfa yield and quality by plant growth-promoting rhizobacteria under saline-alkali conditions. J Sci Food Agric 99:281–289

    CAS  PubMed  Google Scholar 

  27. Lu R (1999) Agricultural chemistry analysis of soil. China Agricultural Science and Technology Press, Beijing

    Google Scholar 

  28. Marques APGC, Pires C, Moreira H, Rangel AOSS, Castro Pml (2010) Assessment of the plant growth promotion abilities of six bacterial isolates using Zea mays as indicator plant. Soil Biol Biochem 42:1229–1235

    CAS  Google Scholar 

  29. Maxton A, Singh P, Masih SA (2017) ACC deaminase-producing bacteria mediated drought and salt tolerance in Capsicum annuum. J Plant Nutr 41(5):574–583

    Google Scholar 

  30. Mayak S, Tirosh T, Glick BR (2004) Plant growth-promoting bacteria confer resistance in tomato plants to salt stress. Plant Physiol Biochem 42(6):565–572

    CAS  PubMed  Google Scholar 

  31. Mukhtar S, Shahid I, Mehnaz S, Malik KA (2017) Assessment of two carrier materials for phosphate solubilizing biofertilizers and their effect on growth of wheat (Triticum aestivum L.). Microbiol Res 205:107–117

    CAS  PubMed  Google Scholar 

  32. Nadeem SM, Ahmad M, Zahir ZA, Javaid A, Ashraf M (2014) The role of mycorrhizae and plant growth promoting rhizobacteria (PGPR) in improving crop productivity under stressful environments. Biotechnol Adv 32:429–448

    PubMed  Google Scholar 

  33. Nadeem SM, Ahmad M, Muhammad Naveed M, Imran M, Ahmad Z, Zahir AZ, Crowley DE (2016) Relationship between in vitro characterization and comparative efficacy of plant growth-promoting rhizobacteria for improving cucumber salt tolerance. Arch Microbiol 198:379–387

    CAS  PubMed  Google Scholar 

  34. Noori F, Etesami H, Zarini HN, Khoshkholgh-Sima NA, Salekdeh GH, Alishahi F (2018) Mining alfalfa (Medicago sativa L.) nodules for salinity tolerant non-rhizobial bacteria to improve growth of alfalfa under salinity stress. Ecotox Environ Safe 162:129–138

    CAS  Google Scholar 

  35. Penrose DM, Glick BR (2003) Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plantarum 118:10–15

    CAS  Google Scholar 

  36. Qin Y, Druzhinina IS, Pan X, Yuan Z (2016) Microbially mediated plant salt tolerance and microbiome-based solutions for saline agriculture. Biotechnol Adv 34:1245–1259

    CAS  PubMed  Google Scholar 

  37. Radhakrishnan R, Hashem A, Abd-Allah EF (2017) Bacillus: a biological tool for crop improvement through bio-molecular changes in adverse environments. Front Physiol 8:667

    PubMed  PubMed Central  Google Scholar 

  38. Reyes-Castillo A, Gerding M, Oyarzúa P, Zagal E, Gerding J, Fischer S (2019) Plant growth-promoting rhizobacteria able to improve NPK availability: selection, identification and effects on tomato growth. Chil J Agr Res 79(3):473–485

    Google Scholar 

  39. Saghafi D, Ghorbanpour M, Lajayer BA (2018) Efficiency of Rhizobium strains as plant growth promoting rhizobacteria on morpho-physiological properties of Brassica napus L. under salinity stress. J Soil Sci Plant Nutr 18(1):253–268

    CAS  Google Scholar 

  40. Samaddar S, Chatterjee P, Choudhury AR, Ahmed S, Sa T (2019) Interactions between Pseudomonas spp. and their role in improving the red pepper plant growth under salinity stress. Microbiol Res 219:66–73

    CAS  PubMed  Google Scholar 

  41. Sapre S, Gontia-Mishra I, Tiwari S (2018) Klebsiella sp. confers enhanced tolerance to salinity and plant growth promotion in oat seedlings (Avena sativa). Microbioll Res 206:25–32

    CAS  Google Scholar 

  42. Saxena AK, Kumar M, Chakdar H, Anuroopa N, Bagyaraj DJ (2020) Bacillus species in soil as a natural resource for plant health and nutrition. J Appl Microbiol 128:1583–1594

    CAS  PubMed  Google Scholar 

  43. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:46–56

    Google Scholar 

  44. Selvakumar G, Kim K, Hu S, Sa T (2014) Effect of salinity on plants and the role of arbuscular mycorrhizal fungi and plant growth-promoting bacteria in alleviation of salt stress. In: Ahmad P, Wani MR (eds) Physiological mechanisms and adaptation strategies in plants under changing environment. Springer, New York, pp 115–144

    Google Scholar 

  45. Sharma SB, Sayyed RZ, Trivedi MH, Gobi TA (2013) Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. SpringerPlus 2:587

    PubMed  PubMed Central  Google Scholar 

  46. Vaishnav A, Choudhary DK (2019) Regulation of drought-responsive gene expression in Glycine max L. merrill is mediated through Pseudomonas simiae strain AU. J Plant Growth Regul 38:333–342

    CAS  Google Scholar 

  47. Wang W, Qiu Z, Tan H, Cao L (2014) Siderophore production by actinobacteria. Biometals 27:623–631

    CAS  PubMed  Google Scholar 

  48. Wang W, Wu Z, He Y, Huang Y, Li X, Ye BC (2018) Plant growth promotion and alleviation of salinity stress in Capsicum annuum L. by Bacillus isolated from saline soil in Xinjiang. Ecotox Environ Safe 164:520–529

    CAS  Google Scholar 

  49. Wu N, Li Z, Wu F, Tang M (2016) Comparative photochemistry activity and antioxidant responses in male and female Populus cathayana cuttings inoculated with arbuscular mycorrhizal fungi under salt. Sci Rep 6:37663

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Yaish MW, Antony I, Glick BR (2015) Isolation and characterization of endophytic plant growth-promoting bacteria from date palm tree (Phoenix dactylifera L.) and their potential role in salinity tolerance. Antonie Van Leeuwenhoek 107:1519–1532

    CAS  PubMed  Google Scholar 

  51. Yamamoto S, Harayama S (1995) PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl Environ Microb 61:1104–1109

    CAS  Google Scholar 

  52. Yasin NA, Akram W, Khan WU, Ahmad SR, Ahmad A, Ali A (2018) Halotolerant plant-growth promoting rhizobacteria modulate gene expression and osmolyte production to improve salinity tolerance and growth in Capsicum annum L. Environ Sci Pollut Res 25(23):23236–23250

    CAS  Google Scholar 

  53. Zhang CS, Kong FY (2014) Isolation and identification of potassium-solubilizing bacteria from tobacco rhizospheric soil and their effect on tobacco plants. Appl Soil Ecol 82:18–25

    Google Scholar 

  54. Zhang WJ, Wang T (2015) Enhanced salt tolerance of alfalfa (Medicago sativa) by rstB gene transformation. Plant Sci 234:110–118

    CAS  PubMed  Google Scholar 

  55. Zhang HH, Li X, Nan X, Sun GY, Sun ml, Cai DJ, Gu SY (2017) Alkalinity and salinity tolerance during seed germination and early seedling stages of three alfalfa (Medicago sativa L.) cultivars. Legume Res 40:853–858

    Google Scholar 

  56. Zhang G, Sun B, Zhao H, Wang X, Zheng C, Xiong K, Ouyan Z, Lu F, Yuan Y (2019) Estimation of greenhouse gas mitigation potential through optimized application of synthetic N, P and K fertilizer to major cereal crops: a case study from China. J Clean Prod 237:117650

    CAS  Google Scholar 

  57. Zhou Y, Tang N, Huang L, Zhao Y, Tang X, Wang K (2018) Effects of salt stress on plant growth, antioxidant capacity, glandular trichome density, and volatile exudates of Schizonepeta tenuifolia Briq. Int J Mol Sci 19(1):252

    PubMed Central  Google Scholar 

Download references

Acknowledgements

We appreciate anonymous reviewers very much for their positive and constructive comments and suggestions on our manuscript. This research was supported by the National Natural Science Foundation of China (31972503), the Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (18KJB210007) and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (XTE1828), China.

Author information

Affiliations

Authors

Contributions

The authors, NG and HJY designed the experiment. ZYZ, HHZ and JL performed the experiments and data analysis. ZYZ wrote the manuscript, and NG edited the manuscript critically and very carefully. HQN, XCC, DL and YC helped perform the analysis with constructive discussions. All authors have read the manuscript and approved the data of the manuscript in its present form.

Corresponding authors

Correspondence to Nan Gao or Hanjie Ying.

Ethics declarations

Conflict of interest

The authors have no conflict of interest regarding the publication of this article.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 118 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhu, Z., Zhang, H., Leng, J. et al. Isolation and characterization of plant growth-promoting rhizobacteria and their effects on the growth of Medicago sativa L. under salinity conditions. Antonie van Leeuwenhoek (2020). https://doi.org/10.1007/s10482-020-01434-1

Download citation

Keywords

  • NaCl
  • Plant growth promoting (PGP)
  • Alfalfa
  • Bacillus sp.
  • Root colonization